An Advanced One-Step RT-LAMP for Rapid Detection of little cherry virus 2 Combined with HTS-based Phylogenomics Reveal Divergent Flowering Cherry Isolates

Abstract

Little cherry virus 2 (LChV-2, genus Ampelovirus) is considered to be the main causal agent of the economically damaging little cherry disease (LChD), which can only be controlled by removal of infected trees. The widespread viral disease of sweet cherry (Prunus avium L.) is affecting the survival of long-standing orchards in North America and Europe, hence the dire need for an early and accurate diagnosis towards a sound disease control strategy. The endemic presence of LChV-2 is mainly confirmed using laborious time-consuming RT-PCR. A rapid RT-LAMP assay targeting a conserved region of the coat protein (CP) was developed and compared with conventional RT-PCR for the specific detection of LChV-2. This affordable assay, combined with a simple RNA extraction, deploys desirable characteristics such as higher ability for faster (<15 min), more analytically sensitive (100-fold) and robust broad-range diagnosis of LChV-2 isolates from sweet cherry, ornamental flowering cherry displaying heterogenous viral etiology and, for the first time, newly-identified potential insect vectors. Moreover, use of Sanger and total RNA High-Throughput Sequencing (HTS) as complementary metaviromics approaches, confirmed the LChV-2 RT-LAMP detection of divergent LChV-2 isolates in new hosts and the relationship of their whole-genome was exhaustively inferred using maximum likelihood phylogenomics. This entails unprecedented critical understanding of a novel evolutionary clade further expanding LChV-2 viral diversity. In conclusion, this highly effective diagnostic platform facilitates strategical support for early in-field testing to reliably prevent dissemination of new LChV-2 outbreaks from propagative plant stocks or newly postulated insect vectors. Validated results and major advantages are herein thoroughly discussed in light of current knowledge ensuing future diagnostic potentials and essential epidemiological considerations to proactively safeguard cherries and Prunus horticultural crop systems from little cherry disease.